Examining the control of differentiation of U937 cells by phorbol esters helps us to understand both the block to differentiation found in human leukemias and how this block may be overcome. Determining the initial cascade of protein kinases which regulate this differentiation pathway defines steps which may be modulated pharmacologically to inhibit leukemic growth. We have shown that bryostatin 1, a non-phorbol ester protein kinase C (pKC) activator, differentiates in vitro peripheral blood cells from patients with CML, AML, and CMML. To further define the pathway by which these agents function, we have examined regulation of the c-Jun protooncogene, a protein that plays a role in regulating gene transcription by binding to DNA and is modified within minutes of PE addition. We have demonstrated that phorbol esters (PEs) and bryostatin 1 induce the transcription of the c-Jun protooncogene and stimulate the phosphorylation of c-Jun protein on serines 63 and 73. We have demonstrated that this phosphorylation is essential to activate the transcriptional stimulatory properties of c-Jun. Further, we have purified a unique protein kinase which stimulates this phosphorylation. Based on these results, it will be the goal of this proposal to determine the cascade of protein kinases which regulate c-Jun function. We will (1) examine the role of specific PKC isoforms in regulating c-Jun phosphorylation and hematopoietic differentiation, (2) determine whether c-Raf-1 is in the PE/bryostatin l cascade of protein kinases, (3) examine the regulation of the c-Jun amino terminal protein kinase during differentiation, and (4) study how phosphorylation of c-Jun mediates its interaction with the transcription machinery. Information gained from these experiments will greatly enhance our knowledge of the early events controlling hematopoietic differentiation in malignant cells and allow us to pinpoint specific areas in which further pharmacologic intervention may be possible.